Unrepaired DNA damage is a prominent feature of vascular cells in diseases that include pulmonary arterial hypertension (PAH). During the current grant cycle, we identified a central role for PPAR?1 in DNA damage sensing and repair that was perturbed in PAH patient pulmonary arterial endothelial cells (PAEC). We observed that PPAR? forms a complex with the DNA damage sensor Mre11, RAD51 and NBS1 (MRN) and with the ubiquitin ligase UBR5. We went on to show that this interaction is necessary for the degradation of ATM interacting protein (ATMIN), thereby permitting phosphorylation of ATM and initiation of the DNA repair process. In PAH PAEC, high interleukin (IL)6 levels are directly related to the phosphorylation of PPAR? at serine 245 and its impaired interaction with MRN and UBR5, causing elevated ATMIN and impaired DNA damage sensing via phosphoATM. In Specific Aim 1, we extend these observations by determining whether high endogenous IL6 levels phosphorylate PPAR? by activating CDK5 and whether phosphorylation of PPAR? at serine 245 disrupts its interaction with UBR5 and MRN. We further determine whether this is a function of loss of BMPR2, the gene mutant in familial PAH and reduced in idiopathic (I) PAH. We also investigate whether sites of unrepaired DNA damage are associated with specific changes in chromatin accessibility and gene regulation that impact cell phenotype. We also determine whether reversal of DNA damage in PAH PAEC, mediated by Nutlin-3-induced p53 and p53-PPAR? dependent genes, restores chromatin accessibility and gene regulation at sites of DNA damage and thus improves PAEC function. To investigate the relationship between cultured PAEC and cells in the intact pulmonary arteries (PA) from PAH and control lungs, we dissociate the cells and incorporate single cell RNA Seq and proximity ligation in situ hybridization (PLISH) on fixed tissue sections to investigate DNA damage in all cells of the vessel wall as this relates to aberrant gene expression. In Specific Aim 2, we use two murine models to study the role of DNA damage in the pathogenesis of pulmonary hypertension (PH). Having shown in mice with EC loss of Bmpr2 that reoxygenation after hypoxia causes DNA damage and persistent PH that is reversed with Nutlin-3, we will use single cell RNA Seq to identify gene expression changes that are linked to damaged and repaired DNA. We will also investigate whether the mouse with DNA damage resulting from loss of ATM in EC has PH, and the PA gene expression changes involved. In Specific Aim 3, we pursue the DNA damage associated with phosphoPPAR? in PAH monocytes and determine whether this is a feature of loss of BMPR2 that is associated with a highly pro-inflammatory pattern of gene expression that can be reversed by roscovitine, the CDK5 inhibitor. Our studies should provide a major inroad into understanding the molecular and functional sequelae of unrepaired DNA in vascular disease and their potential for reversibility of disease.